This invention relates to the fields of somatic cell hybridization, molecular biology, and immunochemistry. More particularly, it concerns a stable human cell line that is a low secretor of IgM and that can be used to prepare human.times.human.times.human triomas, human monoclonal antibodies, and transformants that can be used to express proteins of interest.
Kohler and Milstein, Nature (1975) 256:495-497, pioneered the use of somatic cell hybridization to make continuous hybridomas that produce monoclonal antibodies. Their work used plasmacytomas and lymphocytes of murine origin. Subsequent investigators have applied the techniques of Kohler and Milstein to human cells. Croce et al., Nature (London) (1980) 288:488 and Olsson and Kaplan, Proc. Nat. Acad. Sci. (USA) (1980) 77:5429.
The production of human monoclonal antibodies having a specificity and reproducibility similar to that of mouse monoclonal antibodies has been attempted using various methods. Such methods include, for example, transforming normal human lymphocytes with Epstein-Barr virus (EBV), culturing human B-lymphocytes with antigen, human serum and helper signal producing agents, fusing normal human lymphocytes to human myeloma cells, fusing normal human lymphocytes to an EBV-transformed human lymphoblastoid B-cell line, and fusing human lymphocytes to mouse myeloma or human/rodent heteromyeloma.
Transforming normal human lymphocytes with EBV, such as described in U.S. Pat. No. 4,464,465 and Steinitz, M., Nature (1977) 269:420-422, is cumbersome and therefore not commercially practical. Culturing human B-lymphocytes with antigen, serum and helper signal producing agents, as described in U.S. Pat. No. 4,444,887, requires a relatively complex procedure. Fusing normal human lymphocytes with human myeloma cells, as described by EP No. 44,722 and Cote et al., Proc. Natl. Acad. Sci. USA (1983) 80:2026-2030, is also not practicable due to the limited number of suitable human myeloma cell lines available. Olsson and Kaplan, Proc. Natl. Acad. Sci. USA (1980) 77:5429-5431 describes fusion of a mutant human myeloma cell line of U-266 with lymphoid cells from patients' spleens. Fusing normal human lymphocytes to an EBV-transformed human lymphoblastoid B-cell line suffers in that the capacity of the transformed lines to produce and secrete antibodies typically is such lower than that of myelomas. Examples of such procedures are described further below. Fusing human lymphocytes to mouse or human/rodent myeloma, such as described by U.S. Pat. Nos. 4,634,666 and 4,574,116 and by Kozbor et al., Hybridoma, (1982) 1:323-328, may result in an inherent genetic instability. U.S. Pat. No. 4,634,664 and Ostberg et al., Hybridoma, (1983) 2:361-367 disclose a hybridoma cell line comprising an immortalizing cell fused to a cell producing a predetermined human antibody, the immortalizing cell comprising a xenogeneic hybridoma cell fused from an immortalizing cell and a non-transformed partner cell, the antibody-producing cell being genetically compatible with the non-transformed partner cell. Bron et al., Proc. Natl. Acad. Sci. (1984) 81:3214-3217 describes fusion of an EBV-transformed human B-cell line with a mouse-human heteromyeloma.
Several references describe use of EBV-transformed human B lymphoblastoid cells in producing specific human antibodies. For example, Steinwitz et al., Nature (1977) 269:420 and Luzzanti et al., Nature (1977) 269:419 describe in vitro production of specific human antibodies from such transformed cells. While the EBV transformation allows these cells to be grown continuously, the cells typically lose their ability to secrete Ig in a short period of time.
Several recent references describe using EBV-transformed human lymphoblastoid cell lines as parental tumor partners in fusions with Ig-producing human lymphocytes. European Application No. 82301103.6 published Oct. 13, 1982 describes such a line designated WI-L2-729 HF.sub.2. This line is reported to be a hypoxanthine phosphoribosyl transferase (HPRT)-deficient variant of the WI-L2 line (Levy, J. A. et al., Cancer (1968) 22:517). It is characterized as being nonsecreting, sIgM.sub.K +, cyIgM.sub.K +, and able to grow in serum-free media. Chiorazzi, N. et al., J. Exp. Med. (1982) 156:930-935 describes another EBV-transformed human lymphoblastoid cell line derived from the WI-L2 line. This other line, designated H35.1.1, appears to have different characteristics from the WI-L2-729 HF.sub.2 line. Handley, H. H. et al., Proceedings of the 15th International Leucocyte Culture Conference, Asilomar (1982), p. 267, describes an intermediate parent of the WI-L2-729 HF.sub.2 line, designated UC729-6. UC729-6 is reported to have characteristics common to WI-L2-729 HF.sub.2 and was used as a fusion partner in making Ig-producing human.times.human hybridomas. U.S. Pat. No. 4,451,570 describes preparation of human monoclonal antibodies using a WI-L2 derivative that expresses IgM as the fusion partner. U.S. Pat. No. 4,624,921 discloses a subvariant of the EBV-transformed WI-L2 line, called LTR228, that fuses efficiently with human cells, and copending U.S. Ser. No. 604,069 filed Apr. 26, 1984 discloses fusing LTR228 with a human lymphocyte to produce anti-blood group substance-A antibodies.
Kozbor et al., Proc. Natl. Acad. Sci. USA (1982) 79:6651-6655 describes using an EBV-transformed Ig-producing human lymphocyte as a parental partner in fusion with a 6-thioguanine resistant human lymphoblastoid B-cell line mutagenized and selected for ouabain resistance.
Copending U.S. application Ser. No. 727,821 filed Apr. 26, 1985 and Teng et al., Proc. Natl. Acad. Sci. (USA) (1983) 80:7308-7312 disclose mouse.times.human fusion partner cell lines that can be fused with an antibody-producing human cell line to generate human monoclonal antibodies.
U.S. Pat. No. 4,529,694 discloses fusing human lymphocytes with a human fusion partner that is prepared by fusing human lymphocytes with human myeloma cells. U.S. Pat. No. 4,434,230 discloses hybridomas of human B-lymphocytes and a human non-secretory plasmacytoid continuous cell line. Pickering et al., J. Immunol. (1982) 129:406-412 discloses a human myeloma cell line that does not express immunoglobulin but yields a high frequency of antibody-secreting hybridomas. Kozbor et al. Human Hybridomas and Monoclonal Antibodies, Englemen et al. ed. (Plenum Press, New York, 1985), P. 21-36 disclose fusion partners for producing human monoclonal antibodies including human lymphoblastoid cell lines and non-Ig-secreting partners (p.32). O'Hare et al., Protides of the Biological Fluids, H. Peeters, ed. (Oxford: Pergamon Press, 1983), p.265-268 discloses a new human hybridoma system and alternatives regarding myeloma/lymphoblastoid lines and U-266.
There remains a need in the art for a stable, continuous human cell line that is easily electroporated with DNA for mammalian cell expression, grows rapidly, and secretes a minimal amount of IgM, but when fused to an antibody-producing cell line, secretes adequate amounts of immunoglobulin, including IgG.